The proposed research is a continued study of the involvement of proteins in splicing group I and group II introns. These introns use RNA-catalyzed splicing mechanisms, but require proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. We showed previously that the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) recognizes conserved tRNA-like structural features of the group I intron catalytic core and is by itself sufficient to promote the splicing of different group I introns in vitro. During the current grant period, we obtained a 2.5Angstrom crystal structure of a splicing-compete CYT-18 protein and identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing by acting as an ATPdependent RNA chaperone. In the proposed research, we would continue structural analysis of CYT-18 and its complexes with group I intron RNAs and use CYT-19 as a model system for studying how DExH/D-box proteins mediate RNA conformational changes and acquire specificity for their target RNAs. For group II introns, we have developed an experimental system centered around the mobile Lactococcus lactis L1.LtrB intron, which encodes a reverse transcriptase (LtrA protein) that functions in mobility and as an intron-specific splicing factor (""""""""maturase""""""""). Studies during the current grant period have led to a model in which the LtrA protein promotes splicing by binding first to a high affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding region, and then makes additional contacts with conserved regions of the catalytic core to fold the RNA into the catalytically active structure. In the proposed research, we would continue to study the mechanism of maturase-promoted group II intron splicing, explore the involvement of DExH/D-box proteins in group II intron splicing, and investigate whether maturases can evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.
Specific aims are: (1) To continue structural analysis of the N. crassa CYT-18 protein and its complexes with group I intron RNAs. (2) To continue to investigate how the DEAD-box protein CYT- 19 functions in group I intron splicing and its targeting to CYT-18-dependent group I introns. (3) To continue to investigate the mechanism of maturase-promoted group II intron splicing. (4) To explore the role of DExH/D-box proteins in group II intron splicing. (5) To explore whether some maturases, including chloroplast MatK proteins, can evolve to function in splicing multiple group II introns. This research is intended to provide novel information about how proteins mediate RNA folding and RNA-catalyzed reactions, as well as insight into the evolution of introns and splicing mechanisms, which are fundamentally important for gene expression in higher organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037951-22
Application #
7036608
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
1986-09-01
Project End
2007-07-08
Budget Start
2006-04-01
Budget End
2007-07-08
Support Year
22
Fiscal Year
2006
Total Cost
$479,864
Indirect Cost
Name
University of Texas Austin
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
Mohr, Georg; Kang, Sean Yoon-Seo; Park, Seung Kuk et al. (2018) A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing. J Mol Biol 430:2760-2783
Zubradt, Meghan; Gupta, Paromita; Persad, Sitara et al. (2017) DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat Methods 14:75-82
Stamos, Jennifer L; Lentzsch, Alfred M; Lambowitz, Alan M (2017) Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications. Mol Cell 68:926-939.e4
Bazzini, Ariel A; Del Viso, Florencia; Moreno-Mateos, Miguel A et al. (2016) Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition. EMBO J 35:2087-2103
Burke, James M; Kincaid, Rodney P; Nottingham, Ryan M et al. (2016) DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs. Genes Dev 30:2076-2092
Nottingham, Ryan M; Wu, Douglas C; Qin, Yidan et al. (2016) RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase. RNA 22:597-613
Silas, Sukrit; Mohr, Georg; Sidote, David J et al. (2016) Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. Science 351:aad4234
Qin, Yidan; Yao, Jun; Wu, Douglas C et al. (2016) High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases. RNA 22:111-28
Lamech, Lilian T; Saoji, Maithili; Paukstelis, Paul J et al. (2016) Structural Divergence of the Group I Intron Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases That Function in RNA Splicing. J Biol Chem 291:11911-27
Lambowitz, Alan M; Belfort, Marlene (2015) Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution. Microbiol Spectr 3:

Showing the most recent 10 out of 54 publications